Bone remodelling is a continuous physiological process that occurs in adult skeleton in which bone resorption is followed by new bone formation, maintaining mechanical strength and structure. Bone cells that are responsible for this coupled process include bone-resorbing cells (osteoclasts, which are derived from haematopoietic cells of the monocyte/macrophage lineage) and bone-forming cells (osteoblasts, which are of mesenchymal origin). The bone resorption process is involved in many clinical situations that are relevant to the work of rheumatologists, such as focal bone destruction or erosion in RA and other inflammatory arthritides, and the diffuse bone loss that is encountered in osteoporosis.
Osteoclast activation is a critical cellular process for pathological bone resorption, such as erosions in rheumatoid arthritis (RA) or generalized bone loss. Among many factors triggering excessive osteoclast activity, cytokines such as IL-1 or tumour necrosis factor (TNF)-α play a central role. More recently, the chemokine stromal cell-derived factor-1 (SDF-1) has been shown to promote the chemotactic recruitment, development and survival of human osteoclasts (Wright et al., Bone 36:840-853, 2005; Zannettino et al., Cancer Res 65(5): 1700-1709, 2005; Grassi et al., J. Cell. Physiol. 199:244-251, 2004).
Chemokines are a superfamily of chemoattractant proteins which regulate a variety of biological responses and they promote the recruitment of multiple lineages of leukocytes and lymphocytes to a body organ tissue. Chemokines may be classified into two families according to the relative position of the first two cysteine residues in the protein. In one family, the first two cysteines are separated by one amino acid residue, the CXC chemokines, and in the other family the first two cysteines are adjacent, the CC chemokines. In humans, the genes of the CXC chemokines are clustered on chromosome 4 (with the exception of SDF-1 gene, which has been localized to chromosome 10) and those of the CC chemokines on chromosome 17.
The molecular targets for chemokines are cell surface receptors. One such receptor is CXC chemokine receptor 4 (CXCR4), which is a 7 transmembrane protein, coupled to G1 and was previously called LESTR (Loetscher et al., (1994) J. Biol. Chem, 269: 232-237, 1994), HUMSTR (Federappiel et al., Genomics 16, 707-712, 1993) and Fusin (Feng et al., Science 272: 872-877, 1996). CXCR4 is widely expressed on cells of hemopoietic origin, and is a major co-receptor with CD4+ for human immunodeficiency virus 1 (HIV-1) (Feng at al., Science 272: 872-877, 1996).
Currently, the only known natural ligand for CXCR4 is SDF-1. Stromal cell derived factor-1 alpha (SDF-1 alpha) and stromal cell derived factor-1 beta (SDF-1 beta) are closely related proteins (together referred to herein as SDF-1). The native amino acid sequences of SDF-1 alpha and SDF-1 beta are known, as are the genomic sequences encoding these proteins (U.S. Pat. Nos. 5,563,048 and 5,756,084).
The 3-dimensional crystallographic structure of SDF-1 has been described (Crump et al., EMBO J. 16: 6996-7007, 1997). Structure-activity analysis of SDF-1 indicates that although N-terminal residues 1-8 or 1-9 are involved in receptor binding, the 1-8 and 1-9 peptides alone exhibited no in vitro activity indicative of receptor binding, supporting a reported conclusion that the peptides do not assume the conformation necessary for binding to the receptor. This result was taken to imply that the remainder of the protein scaffold, and/or various consensus receptor binding sites elsewhere in the protein are important for mediating the conformational requirements for N-terminal binding to the receptor (Crump et al., EMBO J. 16: 6996-7007, 1997). Based on these results, a two-site model has been proposed for SDF-1 binding to CXCR4, involving two binding sites in residues 1-17, an N-terminal site and an upstream RFFESH site (Crump at al., EMBO J. 16: 6996-7007, 1997). The two putative binding sites are joined by the CXC motif that characterizes the whole CXC chemokine family. These two putative binding regions have been identified as being important in other CC and CXC chemokines (Crump et al., EMBO J. 16: 6996-7007, 1997). This is consistent with the finding that although N-terminal regions of a wide variety of chemokines are critical for receptor activation, N-terminal peptides of chemokines other than SDF-1 have been reported to lack receptor binding activity and not to be receptor agonists (Crump et al., EMBO J. 16: 6996-7007, 1997).
Postnatal human bone marrow stromal stem cells (BMSSCs) or mesenchymal precursor cells (MPCs) have the capacity to regenerate a hematopoietic-supportive bone marrow organ and associated bone trabecular, when transplanted into immunocompromised mice (Friedenstein et al. Exp Hematol. 6: 440-444, 1978; Kuznetsov et al. J Bone Miner Res. 12: 1335-1347, 1997; Pittenger et al. Science 284: 143-147, 1999; Bianco et al., Stem Cells 19: 180-192, 2001; Gronthos et al. J Cell Sci. 116: 1827-1835, 2003). Recent studies have also reported that BMSSCs are more plastic than first realized, by virtue of their ability to develop into diverse cell lineages such as myelosupportive stroma, osteoblasts, chondrocytes, adipocytes, myoblasts, hepatocytes, cardiomyocytes, and neural cells (Liechty et al. Nat Med. 6: 1282-1286, 2000; Zhao et al. Exp Neurol. 174: 11-20, 2002; Verfaillie et al. Ann N Y Acad Sci. 996: 231-234, 2003). These developments have prompted investigations into the possible use of ex vivo-expanded BMSSC populations for bone regeneration. However, the progress of these studies has largely been restrained because of a lack of understanding of the critical factors that regulate the growth and survival of human multipotential BMSSCs and the eventual development of these cells into bone.